Method of accumulating foreign gene product in plant seed at high level

ABSTRACT

The present inventors succeeded in developing a vector that expresses high levels of a foreign gene in plant seeds by utilizing a 5′-untranslated region of a gene encoding a seed storage protein. The inventors also succeeded in accumulating high levels of a foreign gene product in plant seeds by utilizing a seed storage protein defective mutant as a target for foreign gene transfer.

TECHNICAL FIELD

[0001] The present invention relates to a method for accumulating highlevels of a foreign gene product in plant seeds.

BACKGROUND ART

[0002] Seed storage proteins are conventionally classified into fourgroups of proteins, according to their solubility, i.e., glutelin,globulin, prolamin, and albumin. Rice is different from other grains,such as wheat and maize, in that glutelin is the major seed storageprotein, accounting for about 70 to 80% of the seed storage proteins.The glutelin gene group comprises about 10 genes per one haploid genome,and the genes are divided into two subfamilies, GluA and GluB, whichshow a homology of 60 to 65% at the amino acid sequence level within thecoding region. Each subfamily comprises about 5 genes that have ahomology of 80% or higher at the amino acid sequence level. A glutelingene is specifically expressed and accumulated in the endosperm. Thetissue specificity of glutelin expression is considerably strictlyregulated, and glutelins are not expressed in other tissues, such asleaf and root. The expression of the glutelin gene group, with theexception of GluA-3, is generally coordinated; their mRNA levels showthe following pattern: emerging 5 days after flowering (day 5), reachingmaximum at around day 15, and decreasing thereafter. The GluB-1 gene hasthe strongest promoter activity in the glutelin gene group.

[0003] Rice mutants with decreased amount of accumulated glutelin, i.e.,the major seed storage protein, have been isolated. For example, Iida etal. isolated recessive mutants that lack either one of the acidicsubunits of glutelin, α1, α2, or α3, from a rice breed Koshihikari thatwas irradiated with γ-ray. The phenotypes are respectively regulated bya single recessive gene (i.e., glu1, glu2, or glu3). A mutant strain(α123) that lacks all of α1, α2, and α3 has been also obtained bycrossing the above three mutants (Iida, S. et al., Theor. Appl. Genet.94: 177-183 (1997)).

[0004] LGC-1 (low glutelin content-1) is a mutant selected fromNihonmasari treated with EMS, and has a phenotype with a significantlyreduced level of glutelin (Iida, S. et al., Theor. Appl. Genet. 87:374-378 (1993) ). LGC-1 is further characterized by increased levels ofprolamin and globulin. LGC-1 is dominated by a single dominant gene. Bymapping the defective genes in LGC-1 and the mutants defective of α1,α2, and α3, it was revealed that the mutated protein gene (lgc-1) inLGC-1 and the mutated glutelin gene (glu1) in the mutant lacking α1 arelocalized on the same locus. The results of Southern hybridization usingthe glutelin (GluB) gene as a probe suggested that LGC-1 contained amutation in the GluB gene or in the proximity thereof. According to theresults of Northern blot analysis, comparing the expression level of theGluB gene in the endosperm after about 16 days from head spout in LGC-1and its original breed, Nihonmasari, it was revealed that GluBexpression is markedly decreased in LGC-1.

[0005] In soybean, glycinin is known as a seed storage protein. Glycininis produced as a precursor polypeptide of a size of about 60 kDa whereina signal peptide, an acidic polypeptide, and a basic polypeptide arebound together; the signal peptide is cleaved afterwards. Thereafter, asubunit is formed wherein two kinds of polypeptides that result from acleavage at the Asn-Gly site—i.e., the specific acidic polypeptide (A)and basic polypeptide (B)—are polymerized through disulfide bonds. Sixof these subunits are assembled to form a hexamer, and are stored in theprotein body (PB) The hexamer is also called “11S seed storage protein”,due to its sedimentation coefficient (11S). Glycinin subunits areclassified into group I and group II based on the primary structure oftheir cDNAs and their amino acid sequence homology. To date, subunitsA1aB1b, A1bB2, and A2B1a of group I, and subunits A3B4, and A5A4B3 ofgroup II are known. Six of these subunits are known to be almostrandomly combined in soybean glycinin. Furthermore, a peptide derivedfrom the A1aB1b subunit of soybean glycinin has been reported to havethe ability to bind to bile acid (Shio Makino, The Food Industry 39(24):77-87 (1996)), which suggests that the ability of soybean proteins todecrease the cholesterol level in blood is dependent on the A1aB1bsubunit.

DISCLOSURE OF THE INVENTION

[0006] The present inventors focused on the beneficial physiologicalfunctions of soybean glycinin, such as the cholesterol decreasing effectdescribed above, and have already succeeded in generating rice whereinthe storage protein composition in seeds has been altered by expressingthe A1aB1b gene in the endosperm of the rice seeds (U.S. Pat. No.3,030,339). However, for a desired effect of physiological function toarise from eating the rice, higher levels of expression are necessary.Accordingly, techniques that enable accumulation of higher levels offoreign gene product in rice need to be developed and utilized. Thepresent invention was conducted by taking such requirement into account,and its objective is to provide a method for accumulating high levels offoreign gene product in plant seeds.

[0007] In order to achieve the above objective, the present inventorstried to improve the promoter for expressing high levels of a foreigngene in plant seeds. By examining the promoter region of the rice seedstorage protein glutelin GluB-1 gene, conventional vectors used forexpressing glycinin gene were revealed to incompletely contain the5′-untranslated region of the glutelin gene. The present inventorsfocused on the 5′-untranslated region of the glutelin gene, theimportance of which has not yet been recognized, and examined thewhether the insertion of the 5′-untranslated region into expressionvectors would improve the accumulation level of mRNA. The results showedno improvement in the expression level as compared to the conventionalglycinin gene transductant associated with the insertion of an enhancersequence of a tobacco photosynthesis gene between the GluB-1 genepromoter and the glycinin gene (A1aB1b) (pSaDb). However, insertion ofthe complete 5′-untranslated region of the glutelin (ATG) dramaticallyincreased the accumulation levels of both mRNA and protein.

[0008] Previous studies never considered the maximal capacity of geneexpression (transcription and translation) in expressing a foreign genein plants. Therefore, introduction of a foreign gene was attempted onlyinto plant varieties that were ordinarily used for experiments. Thepresent inventors focused on the “maximal capacity” of plants, andpresumed that foreign gene products might accumulate at higher levelswhen mutants defective in a particular protein were used. Accordingly,the inventors attempted to express and accumulate a foreign geneutilizing mutant plants.

[0009] Several mutants that lack major storage protein, such as LGC-1and α123, are known for rice. The present inventors predicted that theamount of free amino acids available for protein translation in suchseed storage protein defective mutants is larger than in normal plants,since the free amino acids are not utilized for the biosynthesis of thenormally accumulated storage protein. Moreover, the present inventorsconsidered that use of the glutelin promoter in LCG-1 might enablehigher levels of foreign gene expression, since the glutelin geneexpression is suppressed in LGC-1 and thus transcription factors thatare originally used for the expression of glutelin may be utilized forthe foreign gene expression. Thus, the present inventors crossed LGC-1or α123 with 11-5, a glycinin transductant, to introduce the glyciningene into such mutant, and examined the levels of accumulated glycininin their seeds. As a result, they found out that the amount ofaccumulated glycinin protein dramatically increased in both strains ofLGCx11-5 and α123x11-5 as compared with 11-5.

[0010] Thus, the present inventors succeeded in developing a vector thatexpresses high levels of a foreign gene in plant seeds by utilizing the5′-untranslated region of a gene encoding a seed storage protein. Theyalso succeeded in accumulating high levels of a foreign gene product inplant seeds by using a seed storage protein defective mutant as a targetfor gene transfer, and finally accomplished the present invention.

[0011] More specifically, the present invention provides:

[0012] (1) a method for accumulating foreign gene product in plantseeds, comprising the steps of: introducing a foreign gene into a plantthat is defective in endogenous seed storage protein, and expressing theforeign gene in the plant;

[0013] (2) the method according to (1), wherein the foreign gene isintroduced using a vector that comprises the foreign gene operativelyconnected downstream of a promoter which ensures the expression of theforeign gene in plant seeds;

[0014] (3) the method according to (1), wherein the foreign gene isintroduced by crossing with a plant that comprises said foreign gene;

[0015] (4) the method according to (2), wherein a 5′-untranslated regionof a gene encoding a seed storage protein is inserted between theforeign gene and the promoter that ensures the expression of the foreigngene in plant seeds;

[0016] (5) the method according to (4), wherein the 5′-untranslatedregion is a complete one;

[0017] (6) the method according to (4) or (5), wherein the5′-untranslated region is that of a gene encoding a protein selectedfrom the group consisting of glutelin, globulin, prolamin, and albumin;

[0018] (7) the method according to (6), wherein the 5′-untranslatedregion comprises the nucleotide sequence of SEQ ID NO: 1;

[0019] (8) the method according to any one of (1) to (7), wherein thedefective seed storage protein in the plant is selected from the groupconsisting of glutelin, globulin, prolamin, and albumin;

[0020] (9) a transformed plant cell defective in endogenous seed storageprotein into which a foreign gene has been introduced;

[0021] (10) a transformed plant cell defective in endogenous seedstorage protein into which a vector comprising a foreign gene that isoperatively connected downstream of a promoter that ensures theexpression of the foreign gene in plant seeds is introduced;

[0022] (11) the transformed plant cell according to (10), wherein a5′-untranslated region of a gene encoding a seed storage protein isinserted in the expression vector between the foreign gene and thepromoter that ensures the expression of the foreign gene in plant seeds;

[0023] (12) the transformed plant cell according to (11), wherein the5′-untranslated region is a complete one;

[0024] (13) the transformed plant cell according to (11) or (12) whereinthe 5′-untranslated region is that of a gene encoding a protein selectedfrom the group consisting of glutelin, globulin, prolamin, and albumin;

[0025] (14) the transformed plant cell according to (13), wherein the5′-untranslated region comprises the nucleotide sequence of SEQ ID NO:1;

[0026] (15) the transformed plant cell according to any one of (9) to(14), wherein the defective seed storage protein in the plant is aprotein selected from the group consisting of glutelin, globulin,prolamin, and albumin;

[0027] (16) a transgenic plant comprising the transformed plant cellaccording to any one of (9) to (15);

[0028] (17) a vector comprising a promoter that ensures expression inplant seeds and a complete 5′-untranslated region of a gene encoding aseed storage protein that is connected to the promoter;

[0029] (18) the vector according to (17), wherein the 5′-untranslatedregion is that of a gene encoding a protein selected from the groupconsisting of glutelin, globulin, prolamin, and albumin;

[0030] (19) the vector according to (18), wherein the 5′-untranslatedregion of the glutelin gene comprises the nucleotide sequence of SEQ IDNO: 1;

[0031] (20) the vector according to any one of (17) to (19), wherein thepromoter that ensures expression in plant seeds is a promoter of a geneencoding a protein selected from the group consisting of glutelin,globulin, prolamin, and albumin;

[0032] (21) the vector according to any one of (17) to (20), wherein aforeign gene is operatively connected downstream of the 5′-untranslatedregion;

[0033] (22) a transformed plant cell wherein the vector according to(21) is introduced;

[0034] (23) a transgenic plant comprising the transformed plant cellaccording to (22);

[0035] (24) a transgenic plant that is a progeny or clone of thetransformed plant according to (16) or (23); and,

[0036] (25) a breeding material of the transgenic plant according to anyone of (16), (23), and (24).

[0037] The present invention provides a method for accumulating highlevels of a foreign gene product in plant seeds. This method ischaracterized by the use of a mutant plant that is defective inendogenous seed storage protein as a target for expressing the foreigngene. Herein, the term “defective” not only comprises a completedeletion but also a partial deletion. In such plants, the amount of freeamino acids that are available for protein translation is considered tobe larger than in normal plants; this enables efficient accumulation offoreign gene translation product in seeds. There is no particularlimitation on the defective seed storage protein in plants; theinvention includes, for example, glutelin, globulin, prolamin, andalbumin.

[0038] Plants defective in these proteins may be selected from seeds ofplants treated by irradiation, such as with γ-ray, or with amutation-inducing agent, such as EMS and MNU. Mutant plants may beselected by the seed bisection method. Specifically, a seed is crackedin two, and protein is extracted from the endosperm to select seeds withthe desired phenotype. Progenies can be obtained from the embryoscorresponding to the selected endosperm with the desired phenotype.

[0039] Alternatively, plants with a reduced accumulation level of seedstorage protein may. be generated through co-suppression or theantisense method. For co-suppression, a part of a gene encoding a seedstorage protein to be decreased is modified and introduced into plants.In this way, the expressions of genes having a homology higher thab acertain value to the modified gene can be suppressed (for example, inthe above-mentioned LGC-1 mutant, originated from plants irradiated withγ-ray, co-suppression is suggested to have occurred, due to the mutationof the glutelin α1 subunit gene). On the other hand, in the antisensemethod, a DNA encoding an antisense RNA that is complementary to thetranscription product of a gene to be reduced is introduced into plants.

[0040] According to the present invention, known mutants of rice thatlack major storage protein, such as LGC-1 and α123, may be also used.

[0041] Any gene suitable for expression in the seeds of plants may beused as a foreign gene. For example, crops with high additional values,rich in nutrition, having excellent features for processing, and/orfunctioning to maintain and improve health by decreasing the level ofblood cholesterol in human, can be produced using soybean glycinin asthe foreign gene (U.S. Pat. No. 3,030,339). Alternatively, a vaccinegene for passive immune therapy, a modified glutelin gene wherein aphysiologically active peptide is integrated into its variable region,or a useful enzyme gene can be introduced into rice to produce rice withhigh additional value.

[0042] In order to express a foreign gene in plant seeds, a vectorcomprising the foreign gene operatively connected downstream of apromoter that ensures the expression in plant seeds may be favorablyused. Herein, the phrase “operatively connected” means that the foreigngene and the promoter are connected so to express the foreign gene inresponse to the activation of the promoter.

[0043] For example, for expression in rice seeds, the glutelin genepromoter (Takaiwa, F. et al., Plant Mol. Biol. 17: 875-885 (1991)) maybe used as the foreign gene expression promoter. When expressed in beanplants, such as string bean, horse bean, and pea; or oilseed plants,such as peanut, sesame, rapeseed, cottonseed, sunflower, and safflower,the glycinin gene promoter or the promoter of a major storage proteingene of respective plants can be used. For example, the phaseolin genepromoter (Murai, N. et al., Science 222: 476-482 (1983)) and thecruciferin gene promoter (Rodin, J. et al., Plant Mol. Biol. 20: 559-563(1992)) may be used for string bean and rapeseed, respectively. Theabove promoters are just given as examples, and promoters forconstitutive expression, such as the 35S promoter, may be also used.

[0044] It is preferable to insert a 5′-untranslated region of a geneencoding a seed storage protein between the promoter and the foreigngene within a vector for efficient accumulation of the foreign geneproduct in plant seeds. Examples of such a 5′-untranslated regioninclude those of genes encoding glutelin (X54313, Oryza sativa GluA-3gene for glutelin, gi|20207|emb|X54313.1|OSGLUA3[20207]; X54314, O.sativa GluB-1 gene for glutelin, gi|20209|emb|X54314.1|OSGLUB1[20209]),globulin (X62091, LOW MOLECULAR WEIGHT GLOBULIN,gi|5777591|emb|X62091.1|OSLMWG[5777591]), prolamin (D11385, Oryza sativamRNA for prolamin, complete cds, gi|218186|dbj|D11385.1|RICPLM[218186])and albumin (D11431, Rice RA17 gene for allergenic protein, completecds, gi|218194|dbj|D11431.1|RICRA17[218194]; D11432, Rice RA14 gene forallergenic protein, complete cds, gi|218192|dbj|D11432.1|RICRA14[218192]) Especially preferred are 5′-untranslated regions in acomplete form. In the present invention, chimeric 5′-untranslatedregions derived from genes encoding two different seed storage proteinsmay be also used. The complete 5′-untranslated region of the GluB-1 geneis shown in SEQ ID NO: 1.

[0045] A vector wherein a 5′-untranslated region is inserted downstreamof a promoter that ensures the expression in plant seeds and a vectorwherein a foreign gene is further inserted may be constructed using genemanipulation techniques known to those skilled in the art.

[0046] Considering the essence of the present invention, there is nolimitation on plants to derive a plant cell for introducing a vector, solong as it is a seed plant. For example, the plants encompassed by thepresent invention include grains, such as rice, barley, triticum, rye,and maize; beans, such as string bean, horse bean, and pea; and oilseedplants, such as peanut, sesame, rapeseed, cottonseed, sunflower, andsafflower; and so on.

[0047] The forms of plant cell contemplated for introduction of a vectorin the present invention include all kinds of forms that can beregenerated into a plant. For example, the forms encompassed by thepresent invention include cultured cells, protoplast, shoot primordium,polyblasts, hairy roots, and callus, but are not limited thereto. Cellsin a plant are also included in the plant cell of the present invention.

[0048] Methods known to those skilled in the art may be used tointroduce a vector into plant cells. For example, such methods includeindirect transduction, using Agrobacterium tumefaciens or Agrobacteriumrhizogenes (Hiei, Y. et al., Plant J. 6: 271-282 (1994); Takaiwa, F. etal., Plant Sci. 111: 39-49 (1995)); and direct transduction, representedby the electroporation method (Tada, Y. et al., Theor. Appl. Genet. 80:475 (1990)), the polyethylene glycol method (Datta, S. K. et al., PlantMol. Biol. 20: 619-629 (1992)) and the particle bombardment method(Christou, P. et al., Plant J. 2: 275-281 (1992); Fromm, M. E.,Bio/Technology 8: 833-839 (1990)).

[0049] Plants can be produced by regenerating a transformed plant cell.The method for regeneration may differ depending on the type of theplant. However, representative methods include the method of Fujimura etal. (Fujimura, T. et al., Plant Tissue Culture Lett. 2: 74 (1995)), themethod of Armstrong et al. (Armstrong, C. L. and Phillips R. L., CropSci. 28: 363-369 (1988)), and the method of Radke et al. (Radke, S. E.et al., Theor. Appl. Genet. 75: 685-694 (1988)) for rice, maize, andrapeseed, respectively.

[0050] In addition to the above methods, crossing may be used tointroduce a foreign gene into a plant defective in endogenous seedstorage protein. For example, first, a plant harboring the foreign genewithin the genome is generated by introducing the above-mentionedvector. Then, the plant is crossed with a plant that is defective in.endogenous seed storage protein to introduce a foreign gene into theendogenous seed storage protein defective plant.

[0051] Once a transgenic plant, wherein a foreign gene is introducedinto the genome, is obtained, progeny of the plant may be obtained bysexual reproduction. Alternatively, propagative materials (such asseeds, strain, callus, and protoplast) may be obtained from the plantand progeny or clones thereof as the starting material to generate theplant in large quantities. The transgenic plants of the presentinvention can accumulate high levels of a foreign gene product in seedsby expressing the foreign gene. Accordingly, food value, the feature forprocess, health improving function, and such of a seed may beeffectively modified depending on the characteristic of the foreign geneproduct selected for accumulation within the seed. Furthermore,pharmaceutical products and industrial materials may be efficientlymanufactured by accumulating antibody or enzyme in seeds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1 shows the constructs used for examining the effect of a5′-untranslated region (UTR).

[0053]FIG. 2 shows the result of comparison of the measured levels ofglycinin accumulation in the plants transformed with the constructscomprising the 5′-untranslated region of FIG. 1.

[0054]FIG. 3 shows the accumulation and expression of soybean glycininin transgenic rice seeds. (A) shows photographs demonstrating theresults of SDS-PAGE analysis (top) and Northern blot analysis (bottom).(B) shows a chart wherein the results in (A) are quantified andcompared. N indicates the plant comprising a chimeric sequence of theuntranslated regions of the glutelin and glycinin; ATG that comprisingthe complete 5′-untranslated region of the glutelin gene; 11-5 aconventional glycinin gene transductant; and Non-tra a non-transgenicplant.

[0055]FIG. 4 is a photograph depicting the effect of glutelin deficientphenotype on foreign gene product accumulation by SDS-PAGE analysis ofthe endosperm proteins. 11-5 indicates transgenic Matsuyama-miicomprising the glycinin (A1aB1b) gene; LGC indicates LGC-1; and Non-trathe non-transgenic plant.

BEST MODE FOR CARRYING OUT THE INVENTION

[0056] Herein below, the present invention will be specificallydescribed using Examples, but it is not to be construed as being limitedthereto.

EXAMPLE 1 Construction of Soybean Glycinin Expression Vector Using anImproved Promoter, and Generation of Rice Plant Expressing SoybeanGlycinin

[0057] (1) Construction of Chimeric Gene and Gene Transfer

[0058] A cDNA encoding glycinin (A1aB1b) was ligated to GluB-1 genepromoter. Between the cDNA and the promoter, a chimeric sequence (45 bp)of the untranslated regions of glutelin (+1 to 18) and glycinin (−27 toATG) was inserted for N, and the complete 5′-untranslated region (44 bp)of GluB-1 gene for ATG (FIG. 1). As a control, an expression vectorinserted with the 5′-untranslated region of pSaDb, translation enhancersequence of a tobacco photosynthesis gene, was constructed. Theseplasmids comprising these chimeric genes were introduced. into rice(Oryza sativa cv Kitaake) using the Agrobacterium method (Goto, F. etal., Nat. Biotechnol. 17: 282-286 (1999)).

[0059] 11-5 was selected from rice (Oryza sativa cv Matsuyama-mii) towhich a chimeric gene wherein a cDNA encoding glycinin (A1aB1b) wasconnected to GluB-1 gene promoter (−1302 to +18) has been transferred bythe electroporation method.

[0060] (2) Effect of 5′-Untranslated Region of GluB-1 Gene on theExpression of Foreign Gene in Plant Seeds

[0061] A gene was introduced into plants by the Agrobacterium method,and seeds (T1) of the obtained plants were analyzed for the proteinlevel. By comparing N, pSaDb, and ATG, it was revealed that thefrequency of occurrence of plants accumulating high levels of glycininis higher in the order of ATG>N>pSaDb (FIG. 2).

[0062] Next, strains having the highest expression level were selectedfrom each of the N and ATG transgenic strains that accumulated highlevels of glycinin, and were self-fertilized to screen for a homozygote.Then, the levels of mRNA and protein in homozygotes were analyzed asfollows. For RNA analysis, first, RNA was extracted by the SDS-phenolmethod. 12 immature seeds approximately 15 days after flowering werefrozen with liquid nitrogen, and pounded in a mortar into fine powder.Buffer (0.1 M Tris-HCl (pH 9.0), 1% SDS, 0.1 M NaCl, 5 mM EDTA) andphenol-chloroform-isoamyl alcohol (25:24:1) were mixed thereto, andtotal nucleic acid was extracted. The sample was centrifuged to recoverthe supernatant, and was extracted again with phenol-chloroform-isoamylalcohol (25:24:1). Then the total nucleic acid was collected by ethanolprecipitation, and redissolved in distilled water. Then, RNA wasprecipitated in 2M LiCl, and was recovered by centrifugation as thesample. The RNA was electrophoresed on a 1.2% agarose gel, and wastransferred onto a nylon membrane. The prepared membrane was hybridizedwith ³²P-labeled glycinin (A1aB1b) cDNA at 42° C. in 50% (v/v)formamide, 6× SSC, 0.5% (w/v) SDS, and 5× Denhardt's solution. Then, themembrane was washed three times in 2× SSC, 0.1% SDS solution at roomtemperature, and once in 0.1× SSC, 0.1% SDS solution at 55° C. for 20min. For protein analysis, total protein was extracted using 250 μl ofextraction buffer (62.5 mM Tris-HCl (pH 6.8) containing 10% (v/v)glycerol, 0.25% (w/v) SDS, and 5% 2-mercaptoethanol) per 10 mg of matureseed. The extracted protein was treated at 100° C. for 5 min, and thenwas subjected to SDS-PAGE. SDS-PAGE was performed using a 15% (w/v)polyacrylamide gel (acrylamide: N,N′-methylenebisacrylamide=30:0.8).

[0063] As a result, the expression levels of A1aB1b in N and ATG werefound to be 1.43 and 6.56 times, respectively, as much as that in 11-5(FIG. 3). According to a comparison of the protein accumulation level byseparating the acidic subunits of glycinin by SDS-PAGE, the accumulationlevel of A1aB1b in N and ATG were 1.40 and 1.62 times, respectively, asmuch as that in 11-5 (FIG. 3). These results show that the insertion ofa 5′-untranslated region, specifically the complete 5′-untranslatedregion of the GluB-1 gene, between the GluB-1 gene promoter and a cDNAencoding glycinin (A1aB1b) is effective to improve the expression levelof a foreign gene.

EXAMPLE 2 Development of a Technique for Accumulating Foreign GeneProduct at a High Level Using Mutants

[0064] 11-5 (Momma, K. et al., Biosci. Biotechnol. Biochem. 63: 314-318(1999)) was crossed with either LGC-1 (Iida, S. et al., Theor. Appl.Genet. 87: 374-378 (1993)) or α123 (Iida, S. et al., Theor. Appl. Genet.94: 177-183 (1997)), and their F1 seeds were collected. The seeds werecracked in two (the seed bisection method), and endosperm was used forprotein extraction and analysis by SDS-PAGE. Based on the result ofSDS-PAGE analysis, seeds showing an intense band corresponding toglycinin and a weak band for the acidic subunit of glutelin wereselected. By repeating such selection, plants that are homogenous in allphenotypes were obtained.

[0065] The endosperm protein in LGCx11-5 and α123x11-5 were analyzed bySDS-PAGE (FIG. 4). As a result, LGCx11-5 showed the phenotype of LGC-1wherein the band for all 37 to 39 kDa acidic subunits of glutelin becameweak (the total amount of glutelin was decreased to approximately onethird). In contrast, the band corresponding to the acidic subunit of thetransgenic product glycinin was significantly thickened (1.4 fold)compared to the glycinin transductant 11-5. On the other hand, α123x11-5were defective in glutelin acidic subunits α1, α2, or α3, and showed thesame phenotype as α123. In α123x11-5, the band corresponding to theacidic subunits of the transgenic product glycinin was significantlythickened (1.7 fold) compared to the glycinin transductant 11-5.

[0066] Next, the amount of accumulated transgenic product, glycininA1aB1b, was quantified. Specifically, total proteins extracted fromseeds were spotted on nitro cellulose membrane, and were subjected toimmunoblotting using anti-glycinin (A1aB1b) antibody. As a result, theband of the acidic subunit of transgenic product glycinin wassignificantly thickened in seeds of plants that were crossed with LGC-1.Furthermore, a similar result was obtained for those crossed with α123.These results revealed that the addition of the phenotype defective inseed storage protein to a line that accumulate foreign gene product inthe endosperm of seeds enable accumulation of the foreign gene productat a high level.

[0067] Industrial Applicability

[0068] The present invention provides a method for accumulating highlevels of a foreign gene product in plant seeds. The method of thepresent invention may serve as an important fundamental technique fordeveloping useful agricultural products and foods.

1 1 1 44 DNA Oryza sativa 1 tcacatcaat tagcttaagt ttccataagc aagtacaaatagct 44

1. A method for accumulating foreign gene product in plant seeds,comprising the steps of: introducing a foreign gene into a plant that isdefective in endogenous seed storage protein, and expressing the foreigngene in the plant.
 2. The method according to claim 1, wherein theforeign gene is introduced using a vector that comprises the foreigngene operatively connected downstream of a promoter which ensures theexpression of the foreign gene in plant seeds.
 3. The method accordingto claim 1, wherein the foreign gene is introduced by crossing with aplant that comprises said foreign gene.
 4. The method according to claim2, wherein a 5′-untranslated region of a gene encoding a seed storageprotein is inserted between the foreign gene and the promoter thatensures the expression of the foreign gene in plant seeds.
 5. The methodaccording to claim 4, wherein the 5′-untranslated region is a completeone.
 6. The method according to claim 4 or 5, wherein the5′-untranslated region is that of a gene encoding a protein selectedfrom the group consisting of glutelin, globulin, prolamin, and albumin.7. The method according to claim 6, wherein the 5′-untranslated regioncomprises the nucleotide sequence of SEQ ID NO:
 1. 8. The methodaccording to any one of claims 1 to 7, wherein the defective seedstorage protein in the plant is selected from the group consisting ofglutelin, globulin, prolamin, and albumin.
 9. A transformed plant celldefective in endogenous seed storage protein into which a foreign genehas been introduced.
 10. A transformed plant cell defective inendogenous seed storage protein into which a vector comprising a foreigngene that is operatively connected downstream of a promoter that ensuresthe expression of the foreign gene in plant seeds is introduced.
 11. Thetransformed plant cell according to claim 10, wherein a 5′-untranslatedregion of a gene encoding a seed storage protein is inserted in theexpression vector between the foreign gene and the promoter that ensuresthe expression of the foreign gene in plant seeds.
 12. The transformedplant cell according to claim 11, wherein the 5′-untranslated region isa complete one.
 13. The transformed plant cell according to claim 11 or12, wherein the 5′-untranslated region is that of a gene encoding aprotein selected from the group consisting of glutelin, globulin,prolamin, and albumin.
 14. The transformed plant cell according to claim13, wherein the 5′-untranslated region comprises the nucleotide sequenceof SEQ ID NO:
 1. 15. The transformed plant cell according to any one ofclaims 9 to 14, wherein the defective seed storage protein in the plantis a protein selected from the group consisting of glutelin, globulin,prolamin, and albumin.
 16. A transgenic plant comprising the transformedplant cell according to any one of claims 9 to
 15. 17. A vectorcomprising a promoter that ensures expression in plant seeds and acomplete 5′-untranslated region of a gene encoding a seed storageprotein that is connected to the promoter.
 18. The vector according toclaim 17, wherein the 5′-untranslated region is that of a gene encodinga protein selected from the group consisting of glutelin, globulin,prolamin, and albumin.
 19. The vector according to claim 18, wherein the5′-untranslated region of the glutelin gene comprises the nucleotidesequence of SEQ ID NO:
 1. 20. The vector according to any one of claims17 to 19, wherein the promoter that ensures expression in plant seeds isa promoter of a gene encoding a protein selected from the groupconsisting of glutelin, globulin, prolamin, and albumin.
 21. The vectoraccording to any one of claims 17 to 20, wherein a foreign gene isoperatively connected downstream of the 5′-untranslated region.
 22. Atransformed plant cell wherein the vector according to claim 21 isintroduced.
 23. A transgenic plant comprising the transformed plant cellaccording to claim
 22. 24. A transgenic plant that is a progeny or cloneof the transformed plant according to claim 16 or
 23. 25. A breedingmaterial of the transgenic plant according to any one of claims 16, 23,and 24.